Browsing by Subject "Monomeric GTP-Binding Proteins"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item The Regulation of Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2016-04-05) Gulbronson, Connor James; Hansen, Eric J.; Hendrixson, David R.; Norgard, Michael V.; Alto, NealFlagellar biosynthesis is one of the rare processes known to be spatially and numerically regulated in polarly-flagellated bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. FlhG also influences spatial regulation of division, which is essential for viability and is typically regulated by the Min system in most bacteria. However, C. jejuni lacks the Min system, but appears to utilize FlhG and components of the flagellar MS and C ring to influence spatial regulation of division. We utilized a variety imaging techniques to quantify the in vivo effects of mutations in C. jejuni and used purified proteins to assay the in vitro enzymatic activity of FlhG and FlhF (a GTPase) to determine the influence these factors have on both regulation of flagellar biogenesis and spatial regulation of division. We found that unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni instead, other regions of C. jejuni FlhG were discovered to be involved in numerical regulation of flagellar biogenesis. Mutations in the α6 and α7 helices of FlhG were found to influence aspects of FlhG biology, spatial regulation of division, and numerical regulation of flagellar biogenesis. We also found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. In this work, we propose a model in which FlhF in a GTP-bound ('active') state promotes the formation of the MS and C rings at the aflagellated pole after a division event. We then hypothesize that MS and C ring proteins influence FlhG localization to stimulate FlhF GTPase activity and, by extension, numerical regulation of flagellar biogenesis and spatial regulation of division at poles. Although some aspects of this model have yet to be fully tested, our data could potentially be applied in other polar flagellates to gain a better understanding of numerical regulation of flagellar biogenesis and spatial regulation of division in these organisms.Item The Small GTPase Rheb Is Required for Spermatogenesis but not Oogenesis(2013-10-01) Baker, Michael David; Lum, Lawrence; Castrillon, Diego H.; Brekken, Rolf A.; Amatruda, James F.The process of germ cell development is under the tight control of various signaling pathways among which the PI3K-PKB-mTOR pathway is of critical importance. Previous studies have demonstrated sex-specific roles for several components of this pathway. In the current study I aimed to evaluate the role of Rheb, a member of the small GTPase superfamily and a critical component for mTORC1 activation, in male and female gametogenesis. The function of Rheb in development and the nervous system has been extensively studied, but little was known about its role in the germline. I have exploited genetic approaches in the mouse to study the role of Rheb in the germline and have identified an essential role in spermatogenesis. Conditional knockout (cKO) of Rheb in the male germline resulted in severe oligoasthenoteratozoospermia and male sterility. More detailed phenotypic analyses uncovered an age-dependent meiotic progression defect combined with subsequent abnormalities in spermiogenesis as evidenced by abnormal sperm morphology. In the female, however, germ-cell specific inactivation of Rheb was not associated with any discernible abnormality; these cKO mice were fertile with morphologically unremarkable ovaries, normal primordial follicle formation, and subsequent follicle maturation. The absence of an abnormal ovarian phenotype is striking given previous studies demonstrating a critical role for the mTORC1 pathway in the maintenance of primordial follicle pool. In conclusion, our findings demonstrate an essential role of Rheb in diverse aspects of spermatogenesis but suggest the existence of functionally-redundant factors that can compensate for Rheb deficiency within oocytes.